Identification of Putative New Splicing Targets for ETR-3 Using Sequences Identified by Systematic Evolution of Ligands by Exponential Enrichment

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 2005 Jan 20

PMID 15657417

Citations 63

Authors

Nuno Andre Faustino

Thomas A Cooper

Affiliations

Soon will be listed here.

Abstract

ETR-3 (also know as BRUNOL3, NAPOR, and CUGBP2) is one of six members of the CELF (CUG-BP1- and ETR-3-like factor) family of splicing regulators. ETR-3 regulates splicing by direct binding to the pre-mRNA. We performed systematic evolution of ligands by exponential enrichment (SELEX) to identify the preferred binding sequence of ETR-3. After five rounds of SELEX, ETR-3 selected UG-rich sequences, in particular UG repeats and UGUU motifs. Either of these selected motifs was able to restore ETR-3 binding and responsiveness to a nonresponsive splicing reporter in vivo. Moreover, this effect was not specific to ETR-3 since minigenes containing either of the two motifs were responsive to two other CELF proteins (CUG-BP1 and CELF4), indicating that different members of the CELF family can mediate their effects via a common binding site. Using the SELEX-identified motifs to search the human genome, we identified several possible new ETR-3 targets. We created minigenes for two of these genes, the CFTR and MTMR1 genes, and confirmed that ETR-3 regulates their splicing patterns. For the CFTR minigene this regulation was demonstrated to be dependent on the presence of the putative binding site identified in our screen. These results validate this approach to search for new targets for RNA processing proteins.

Citing Articles

CELF1 represses Doublesex1 expression via its 5' UTR in the crustacean Daphnia magna.

Tirta Y, Adachi S, Perez C, Adhitama N, Nong Q, Natsume T PLoS One. 2022; 17(10):e0275526.

PMID: 36240182 PMC: 9565731. DOI: 10.1371/journal.pone.0275526.

Investigating the ACE2 polymorphisms in COVID-19 susceptibility: An in silico analysis.

Pouladi N, Abdolahi S Mol Genet Genomic Med. 2021; 9(6):e1672.

PMID: 33818000 PMC: 8222831. DOI: 10.1002/mgg3.1672.

CELF2 regulates the species-specific alternative splicing of TREM2.

Yanaizu M, Washizu C, Nukina N, Satoh J, Kino Y Sci Rep. 2020; 10(1):17995.

PMID: 33093587 PMC: 7582162. DOI: 10.1038/s41598-020-75057-x.

Alternative splicing regulation of doublesex gene by RNA-binding proteins in the silkworm Bombyx mori.

Zheng Z, Sun X, Zhang B, Pu J, Jiang Z, Li M RNA Biol. 2019; 16(6):809-820.

PMID: 30836863 PMC: 6546364. DOI: 10.1080/15476286.2019.1590177.

Myotonic Dystrophy and Developmental Regulation of RNA Processing.

Thomas J, Oliveira R, Sznajder L, Swanson M Compr Physiol. 2018; 8(2):509-553.

PMID: 29687899 PMC: 11323716. DOI: 10.1002/cphy.c170002.

References

Niksic M, Romano M, Buratti E, Pagani F, Baralle F . Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9. Hum Mol Genet. 1999; 8(13):2339-49. DOI: 10.1093/hmg/8.13.2339. View

Savkur R, Philips A, Cooper T . Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet. 2001; 29(1):40-7. DOI: 10.1038/ng704. View

Savkur R, Philips A, Cooper T, Dalton J, Moseley M, Ranum L . Insulin receptor splicing alteration in myotonic dystrophy type 2. Am J Hum Genet. 2004; 74(6):1309-13. PMC: 1182097. DOI: 10.1086/421528. View

Pagani F, Buratti E, Stuani C, Romano M, Zuccato E, Niksic M . Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element. J Biol Chem. 2000; 275(28):21041-7. DOI: 10.1074/jbc.M910165199. View

Jensen K, Musunuru K, Lewis H, Burley S, Darnell R . The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain. Proc Natl Acad Sci U S A. 2000; 97(11):5740-5. PMC: 18503. DOI: 10.1073/pnas.090553997. View

Bolino A, Muglia M, Conforti F, Leguern E, Salih M, Georgiou D . Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2. Nat Genet. 2000; 25(1):17-9. DOI: 10.1038/75542. View

Ladd A, Charlet N, Cooper T . The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol. 2001; 21(4):1285-96. PMC: 99581. DOI: 10.1128/MCB.21.4.1285-1296.2001. View

Iakova P, Wang G, Timchenko L, Michalak M, Pereira-Smith O, Smith J . Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate. EMBO J. 2004; 23(2):406-17. PMC: 1271759. DOI: 10.1038/sj.emboj.7600052. View

White E, Ruley H . PUM2, a novel murine puf protein, and its consensus RNA-binding site. RNA. 2002; 7(12):1855-66. PMC: 1370223. View

10.

Larriba S, Bassas L, Gimenez J, Ramos M, Segura A, Nunes V . Testicular CFTR splice variants in patients with congenital absence of the vas deferens. Hum Mol Genet. 1998; 7(11):1739-43. DOI: 10.1093/hmg/7.11.1739. View

11.

Cuppens H, Lin W, Jaspers M, Costes B, Teng H, Vankeerberghen A . Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J Clin Invest. 1998; 101(2):487-96. PMC: 508589. DOI: 10.1172/JCI639. View

12.

Laporte J, Blondeau F, Buj-Bello A, Mandel J . The myotubularin family: from genetic disease to phosphoinositide metabolism. Trends Genet. 2001; 17(4):221-8. DOI: 10.1016/s0168-9525(01)02245-4. View

13.

Singh G, Charlet-B N, Han J, Cooper T . ETR-3 and CELF4 protein domains required for RNA binding and splicing activity in vivo. Nucleic Acids Res. 2004; 32(3):1232-41. PMC: 373409. DOI: 10.1093/nar/gkh275. View

14.

Buj-Bello A, Furling D, Tronchere H, Laporte J, Lerouge T, Butler-Browne G . Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells. Hum Mol Genet. 2002; 11(19):2297-307. DOI: 10.1093/hmg/11.19.2297. View

15.

Zhang W, Liu H, Han K, Grabowski P . Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR. RNA. 2002; 8(5):671-85. PMC: 1370287. DOI: 10.1017/s1355838202027036. View

16.

Laporte J, Hu L, Kretz C, Mandel J, Kioschis P, Coy J . A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast. Nat Genet. 1996; 13(2):175-82. DOI: 10.1038/ng0696-175. View

17.

Simonsen A, Wurmser A, Emr S, Stenmark H . The role of phosphoinositides in membrane transport. Curr Opin Cell Biol. 2001; 13(4):485-92. DOI: 10.1016/s0955-0674(00)00240-4. View

18.

Liu H, Zhang M, Krainer A . Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 1998; 12(13):1998-2012. PMC: 316967. DOI: 10.1101/gad.12.13.1998. View

19.

Mankodi A, Urbinati C, Yuan Q, Moxley R, Sansone V, Krym M . Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum Mol Genet. 2001; 10(19):2165-70. DOI: 10.1093/hmg/10.19.2165. View

20.

Kino Y, Mori D, Oma Y, Takeshita Y, Sasagawa N, Ishiura S . Muscleblind protein, MBNL1/EXP, binds specifically to CHHG repeats. Hum Mol Genet. 2004; 13(5):495-507. DOI: 10.1093/hmg/ddh056. View